This project is directed to a molecular characterization of the coupling mechanism of catalysis and Ca2+ transport in the sarcoplasmic reticulum ATPase and its isoforms. Characterization will be sought in terms of protein structure and function. The following experimental approaches will be taken in pursuit of the general aim: 1) Chemical derivatization of selected amino acid residues of the sarcoplasmic reticulum ATPase purified from muscle, determination of the residues undergoing derivatization, characterization of the functional consequences of derivatization, and spectroscopic studies of chromophoric labels. 2) Transient and stable expression of cDNA encoding the Ca2+ -ATPase, recovery of functionally competent protein from transfected cells, induction of site directed mutations, and functional evaluation of unmutated and mutated forms of the ATPase. 3) Kinetic and equilibrium characterization of the partial reactions of the catalytic and transport cycle using native or derivatized ATPase obtained from muscle, as well as unmutated and mutated enzyme expressed in cell cultures. Evaluation of inhibitors and regulatory mechanisms. The findings originating from this project are expected to clarify basic aspects of transduction mechanisms in proteins, as well as aspects of intracellular Ca2+ regulation. The latter relates directly contractile activation and relaxation in cardiac and skeletal muscle.